Analgesic and potentiated photothermal therapy with ropivacaine-loaded hydrogels

Rationale: Tumor ablation can cause severe pain to patients, but there is no satisfactory means of analgesia available. In addition, recurrence of residual tumors due to incomplete ablation threatens patient safety. Photothermal therapy (PTT), a promising approach for tumor ablation, also faces the aforementioned problems. Therefore, developing novel photothermal agents that can efficiently relieve PTT-associated pain and potentiate the PTT efficacy are urgently needed. Methods: The Pluronic F127 hydrogel doped with indocyanine green (ICG) was served as photothermal agent for PTT. Mouse model that inoculation of tumor near the sciatic nerve was constructed to assess the PTT-evoked pain. Subcutaneous and sciatic nerve vicinal tumor-bearing mice were used to test the efficacy of PTT. Results: PTT-evoked pain depends on an increase in tumor temperature and is accompanied by the activation of TRPV1. A simple introduction of local anesthetic (LA) ropivacaine into ICG-loaded hydrogels relieves PTT-induced pain and exerts long-lasting analgesia compared with opioid analgesia. More interestingly, ropivacaine upregulates major histocompatibility complex class I (MHC-I) in tumor cells by impairing autophagy. Therefore, a hydrogel co-doped with ropivacaine, TLR7 agonist imiquimod and ICG was rationally designed. In the hydrogel system, imiquimod primes tumor-specific CD8+ T cells through promoting DCs maturation, and ropivacaine facilitates tumor cells recognition by primed CD8+ T cells through upregulating MHC-I. Consequently, the hydrogel maximumly increases CD8+ T cells infiltration into tumor and potentiates PTT efficacy. Conclusion: This study for the first time provides an LA-dopped photothermal agents for painless PTT and innovatively proposes that a LA can be used as an immunomodulator to potentiate the PTT efficacy.


In vitro hydrogel degradation
The in vitro degradation of hydrogels was determined by a weight remaining (%) experiment described by previous literature [1]. Briefly, samples of 500 μL of PFIR hydrogel formulations were added into glass vials and placed at 37 °C. After gelation, the original weight of each hydrogel formulation was recorded as W0. 1 mL of preequilibrated PBS (pH 7.4) was gently laid over the surface of the hydrogels and incubated in a metal bath at 37 °C. The weight of the remaining hydrogel samples (Wt) was recorded at regular time intervals (1,3,6,9,12,24,36, and 48 h) after removing the supernatant. The in vitro degradation assay was performed in triplicate. The weight remaining (%) was calculated as: ℎ (%) = / 0 × 100%.

Degradation of hydrogel in vivo
100 μL of PFIR hydrogel subcutaneously injected into the right flank of BALB/c mice.
At indicated timepoints, the mice were sacrificed, and the residual hydrogel in the subcutaneous layer was photographed.
Twenty-four hours after laser irradiation, cell viability were assayed using a CCK-8 staining were observed at a confocal fluorescence microscope (LSM800, Zeiss).

Detection of the relief of cancer pain by PFR
Mice were anesthetized with isoflurane. One million 4T1 cells in 100 μL of sterile PBS were injected into the muscular tissue in the immediate vicinity of the nerve near the trochanter [2]. Ten days after inoculation, the paw was observed to curl up, indicating the successful establishment of a mouse model of spontaneous cancer pain. To test the analgesic effect, 100 μL 10% (w/v) free ropivacaine or PFR containing 10% ropivacaine (w/ v) was injected into the tumor near the sciatic nerve [3]. Then the posture of mouse paw was observed and mechanical pain was tested at indicated timepoint after administration.

Results
Figure S1                        PFI, PFIM, PFIR or PFIRM in 500 μL DMEM culture medium, cells were subjected to laser irradiation at 1.2 W/cm 2 for 5 min. Twenty-four hours after irradiation, cells were stained, and the concentration of ATP or HMGB-1 in culture medium were quantified. The assay was repeated three times. Data represents mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001. 'NS' to indicate non-significance.